The invention was based on the object of finding novel compounds having valuable properties, in particular those which can be used for the preparation of medicaments.
The present invention relates to compounds and the use thereof for the modulation, in particular for the inhibition, of the activity or function of the phosphoinositide 3′-OH kinase family (hereinafter PI3 kinases), advantageously PI3Kα, PI3Kδ, PI3Kβ and/or PI3Kγ. The present invention advantageously relates to the use of pyridinylimidazolone derivatives in the treatment of one or more disease states selected from: autoimmune disorders, inflammatory diseases, cardiovascular diseases, neurodegenerative diseases, allergy, asthma, pancreatitis, multiorgan failure, kidney diseases, blood platelet aggregation, cancer, sperm motility, transplant rejection, graft rejection and lung injuries.
Cell membranes provide a large store of secondary messengers that can be enlisted in a variety of signal transduction pathways. As regards the function and regulation of effector enzymes in phospholipid signalling pathways, these enzymes generate secondary messengers from the membrane phospholipid pools. Class I PI3 kinases (for example PI3Kα) are dual-specificity kinase enzymes, i.e. they exhibit both lipid kinase activity (phosphorylation of phosphoinositides) and protein kinase activity, shown to be capable of phosphorylation of protein as substrate, including autophosphorylation as intramolecular regulatory mechanism. These enzymes of phospholipid signalling are activated by various extracellular signals, such as growth factors, mitogens, integrins (cell-cell interactions), hormones, cytokines, viruses, and neurotransmitters, as described in Scheme I below, and also by intracellular regulation by other signaling molecules (cross-talk, where the original signal can activate some parallel pathways, which in a second step transmit signals to PI3Ks by intracellular signaling events), such as, for example, small GTPases, kinases, or phosphatases. Intracellular regulation can also occur as a result of aberrant expression or lack of expression of cellular oncogenes or tumour suppressors. The intracellular inositol phopholipid (phosphoinositide) signaling pathways begin with activation of signaling molecules (extracellular ligands, stimuli, receptor dimerisation, transactivation by a heterologous receptor (for example receptor tyrosine kinase) and with the recruitment and activation of PI3K, including the involvement of G protein-linked transmembrane receptor integrated into the plasma membrane.
PI3K converts the membrane phospholipid PI(4,5)P2 into PI(3,4,5)P3, which functions as secondary messenger. PI and PI(4)P are likewise substrates of PI3K and can be phosphorylated and converted into PI3P and PI(3,4)P2, respectively. In addition, these phosphoinositides can be converted into other phosphoinositides by 5′-specific and 3′-specific phosphatases, meaning that PI3K enzyme activity results either directly or indirectly in the generation of two 3′-phosphoinositide subtypes which function as secondary messengers in intracellular signal transduction pathways (Trends Biochem. Sci. 22(7) pp. 267-72 (1997) by Vanhaesebroeck et al; Chem. Rev. 101(8) pp. 2365-80 (2001) by Leslie et al (2001); Annu. Rev. Cell. Dev. Biol. 17p, 615-75 (2001) by Katso et al. and Cell. Mol. Life Sci. 59(5) pp. 761-79 (2002) by Toker et al.). Multiple PI3K isoforms categorised by their catalytic subunits, their regulation by corresponding regulatory subunits, expression patterns and signal-specific functions (p110α, β, δ and γ) perform this enzyme reaction (Exp. Cell. Res. 25 (1) pp. 239-54 (1999) by Vanhaesebroeck and Katso et al., 2001, see above).
The closely related isoforms p110α and β are expressed ubiquitously, while δ and γ are expressed more specifically in the haematopoietic cell system, in the smooth muscle cells, myocytes and endothelial cells (Trends Biochem. Sci. 22(7) pp. 267-72 (1997) by Vanhaesebroeck et al.). Their expression can also be regulated in an inducible manner depending on the cellular tissue type and stimuli as well as in accordance with the particular disease. The inducibility of protein expression includes protein synthesis as well as protein stabilisation, which is partly regulated by association with regulatory subunits.
To date, eight mammalian PI3Ks have been identified, divided into 3 main classes (I, II and III) on the basis of sequence homology, structure, binding partners, mode of activation, and substrate preference. In vitro, class I PI3Ks are able to phosphorylate phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to give phosphatidylinositol 3-phosphate (PI3P), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2, and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3, respectively. Class II PI3Ks phosphorylate PI and phosphatidylinositol 4-phosphate. Class III PI3Ks can only phosphorylate PI (Vanhaesebroeck et al., 1997, see above; Vanhaesebroeck et al., 1999, see above, and Leslie et al, 2001, see above).

As illustrated in Scheme I above, phosphoinositide 3-kinases (PI3Ks) phosphorylate the hydroxyl of the third carbon atom on the inositol ring. The phosphorylation of phosphoinositides which converts Ptdlns into 3,4,5-triphosphate (Ptdlns(3,4,5)P3), Ptdlns(3,4)P2 and Ptdlns(3)P produces secondary messengers for various signal transduction pathways, as are essential, inter alia, for cell proliferation, cell differentiation, cell growth, cell size, cell survival, apoptosis, adhesion, cell mobility, cell migration, chemotaxis, invasion, cytoskeletal rearrangement, cell shape changes, vesicle trafficking and metabolic pathway (Katso et al, 2001, see above, and Mol. Med. Today 6(9) pp. 347-57 (2000) by Stein). G protein-coupled receptors mediate phosphoinositide 3′-OH kinase activation via small GTPases, such as Gβγ and Ras, and consequently PI3K signaling plays a central role in the development and coordination of cell polarity and dynamic organisation of the cytoskeleton—which together provide the driving force for cell movement.
Chemotaxis—the directed movement of cells in the direction of a concentration gradient of chemical attractants, which are also called chemokines, is also involved in many important diseases, such as inflammation/autoimmunity, neurodegeneration, angiogenesis, invasion/metastasis and wound healing (Immunol. Today 21(6) pp. 260-4 (2000) by Wyman et al.; Science 287(5455) pp. 1049-53 (2000) by Hirsch et al.; FASEB J. 15(11) pp. 2019-21 (2001) by Hirsch et al., and Nat. Immunol. 2(2) pp. 108-15 (2001) by Gerard et al.).
Advances using genetic approaches and pharmacological tools have provided insights into signalling and molecular pathways which promote chemotaxis in response to chemical attractant-activated, G protein-coupled sensors. PI3 kinase, which is responsible for the generation of these phosphorylated signaling products, was originally identified as an activity which is associated with viral oncoproteins and growth factor tyrosine kinases which phosphorylate phosphatidylinositol (PI) and its phosphorylated derivatives at the 3′-hydroxyl of the inositol ring (Panayotou et al., Trends Cell Biol. 2 pp. 358-60 (1992)). However, more recent biochemical studies have shown that class I PI3 kinases (for example class IB isoform PI3Kγ) are dual-specificity kinase enzymes, which means that they exhibit both lipid kinase activity and protein kinase activity, shown to be capable of phosphorylation of other proteins as substrates, as well as autophosphorylation as an intramolecular regulatory mechanism.
PI3 kinase activation is therefore probably involved in various cellular responses, including cell growth, differentiation and apoptosis (Parker et al., Current Biology, 5 pp. 577-99 (1995); Yao et al., Science, 267 pp. 2003-05 (1995)). PI3 kinases appear to be involved in a number of aspects of leukocyte activation. A p85-associated PI3 kinase activity has been shown to associate physically with the cytoplasmic domain of CD28, which is an important co-stimulatory molecule for the activation of T cells by antigen (Pages et al., Nature, 369 pp. 327-29 (1994); Rudd, Immunity 4 pp. 527-34 (1996)). Activation of T cells by CD28 lowers the threshold for activation by antigen and increases the magnitude and duration of the proliferative response. These effects are accompanied by increases in the transcription of a number of genes, such as, inter alia, interleukin-2 (IL2), an important T cell growth factor (Fraser et al., Science 251 pp. 313-16 (1991)). If CD28 is mutated in such a way that it can no longer interact with PI3 kinase, initiation of IL-2 production fails, which suggests a crucial role for PI3 kinase in T cell activation. PI3Kγ has been identified as a promoter of G-β-γ-dependent regulation of JNK activity, and G-β-γ are subunits of heterotrimeric G proteins (Lopez-Ilasaca et al, J. Biol. Chem. 273(5) pp. 2505-8 (1998)). Cellular processes in which PI3Ks play an essential role include suppression of apoptosis, reorganisation of the actin skeleton, cardiac myocyte growth, glycogen synthase stimulation by insulin, TNFα-promoted neutrophil priming and superoxide generation, and leukocyte migration and adhesion to endothelial cells.
Laffargue et al., Immunity 16(3) pp. 441-51 (2002), have described that PI3Kγ relays inflammatory signals via various G(i)-coupled receptors and that it is crucial for mast cell function, stimuli in connection with leukocytes, and immunology, including cytokines, chemokines, adenosines, antibodies, integrins, aggregation factors, growth factors, viruses or hormones (J. Cell. Sci. 114(Pt 16) pp. 2903-10 (2001) by Lawlor et al.; Laffargue et al., 2002, see above, and Curr. Opinion Cell Biol. 14(2) pp. 203-13 (2002) by Stephens et al.).
Specific inhibitors against individual members of a family of enzymes provide invaluable tools for deciphering the functions of each enzyme. Two compounds, LY294002 and wortmannin (see below), have been widely used as PI3 kinase inhibitors. These compounds are non-specific PI3K inhibitors, since they do not distinguish between the four members of class I PI3 kinases. For example, the IC50 values of wortmannin against each of the various class I PI3 kinases are in the range from 1 to 10 nM. Correspondingly, the IC50 values of LY294002 against each of these PI3 kinases are about 15 to 20 μM (Fruman et al., Ann. Rev. Biochem., 67, pp. 481-507 (1998)), in addition it has IC50 values of 5-10 μM on CK2 protein kinase and a slight inhibitory activity on phospholipases. Wortmannin is a fungal metabolite which irreversibly inhibits PI3K activity by bonding covalently to the catalytic domain of this enzyme. The inhibition of PI3K activity by wortmanin eliminates the subsequent cellular response to the extracellular factor. For example, neutrophils respond to the chemokine fMet-Leu-Phe (fMLP) by stimulation of PI3K and synthesis of Ptdlns (3, 4, 5)P3. This synthesis correlates with activation of the respiratory burst which is involved in the destruction of the neutrophils of invading micro-organisms. Treatment of neutrophils with wortmannin prevents the fMLP-induced respiratory burst response (Thelen et al., Proc. Natl. Acad. Sci. USA, 91, pp. 4960-64 (1994)). Indeed, these experiments with wortmannin, as well as other experimental evidence, show that PI3K activity in cells of haematopoietic lineage, in particular neutrophils, monocytes and other types of leukocytes, are involved in many of the non-memory immune response associated with acute and chronic inflammation.

Based on studies with wortmannin, there is evidence that PI3 kinase function is also necessary for some aspects of leukocyte signalling by G protein-coupled receptors (Thelen et al., 1994, see above). In addition, it has been shown that wortmannin and LY294002 block neutrophil migration and super-oxide release. Carboxygenase-inhibiting benzofuran derivatives are disclosed by John M. Janusz et al., in J. Med. Chem. 1998; Vol. 41, No. 18.
It is now well understood that deregulation of oncogenes and tumour-suppressor genes contributes to the formation of malignant tumours, for example by increasing cell growth and proliferation or increased cell survival. It is now also known that signalling pathways promoted by the PI3K family play a central role in a number of cell processes, such as, inter alia, in proliferation and survival, and deregulation of these pathways is a causative factor in a broad spectrum of human cancer diseases and other diseases (Katso et al., Annual Rev. Cell Dev. Biol, 2001, 17: 615-617, and Foster et al, J. Cell Science. 2003, U6: 3037-3040).
Class I PI3K is a heterodimer consisting of a catalytic p110 subunit and a regulatory subunit, and the family is further divided into class Ia and class Ib enzymes on the basis of the regulatory partners and the regulation mechanisms. Class Ia enzymes consist of three different catalytic subunits (p110α, p110β, and p110δ), which dimerise with five different regulatory subunits (p85α, p55α, p50α, p85β and p55γ), where all catalytic subunits are able to interact with all regulatory subunits to form various heterodimers. Class Ia PI3Ks are generally activated in response to growth factor stimulation of receptor tyrosine kinases via interaction of the regulatory SH2 domain subunit with specific phosphotyrosine residues of the activated receptor or adaptor proteins, such as IRS-1. Small GTPases (for example ras) are likewise involved in the activation of PI3K together with receptor tyrosine kinase activation. Both p110α and p110β are constitutively involved in all cell types, whereas p110δ expression is more restricted to leukocyte populations and some epithelial cells. By contrast, the only class Ib enzyme consists of a catalytic p110γ subunit, which interacts with a regulatory p101 subunit. In addition, the class Ib enzyme is activated by G protein-coupled receptor (GPCR) systems, and its expression appears to be limited to leukocytes.
There is now clear evidence showing that class Ia PI3K enzymes contribute to tumorigenesis in a large number of human cancer diseases, either directly or indirectly (Vivanco and Sawyers, Nature Reviews Cancer, 2002, 2, 489-501). For example, the p110α subunit is amplified in some tumours, such as, for example, in ovarian tumours (Shayesteh, et al., Nature Genetics, 1999, 21: 99-102) and cervix (Ma et al, Oncogene, 2000, 19: 2739-2744). Recently, activating mutations in p110α (PIK3CA gene) have been associated with various other tumours, such as, for example, colon and breast and lung tumours (Samuels, et al., Science, 2004, 304, 554). Tumour-related mutations in p85α have likewise been identified in cancer diseases, such as ovarian and colon cancer (Philp et al., Cancer Research, 2001, 61, 7426-7429). Besides direct effects, activation of class I PI3Ks is probably involved in tumorigenic events occurring upstream of signalling pathways, for example by means of ligand-dependent or ligand-independent activation of receptor tyrosine kinases, GPCR systems or integrins (Vara et al., Cancer Treatment Reviews, 2004, 30, 193-204). Examples of such upstream signalling pathways include overexpression of the receptor tyrosine kinase Erb2 in a number of tumours which lead to activation of PI3K-promoted pathways (Harari et al., Oncogene, 2000, Jj), 6102-6114) and overexpression of the oncogene Ras (Kauffmann-Zeh et al., Nature, 1997, 385, 544-548). In addition, class Ia PI3Ks may con-tribute indirectly to tumorigenesis caused by various downstream signalling events. For example, the loss of function of the PTEN tumour-suppressor phosphatase which catalyses the conversion of PI(3,4,5,)P3 back to PI(4,5)P2 is associated with a very broad range of tumours via deregulation of the PI3K-promoted production of PI(3,4,5)P3 (Simpson and Parsons, Exp. Cell Res., 2001, 264, 29-41). In addition, the increase in the effects of other PI3K-promoted signalling events probably contributes to a number of cancer diseases, for example by activation of AKT (Nicholson and Andeson, Cellular Signaling, 2002, 14, 381-395).
Besides a role in the promotion of proliferative and survival signalling in tumour cells, there is good evidence that class I PI3K enzymes also contribute to tumorigenesis via their function in tumour-associated stromal cells. PI3K signalling is known to play an important role in the promotion of angiogenic events in endothelial cells in response to pro-angiogenic factors, such as VEGF (abid et al., Arterioscler. Thromb. Vasc. Biol., 2004, 24, 294-300). Since class I PI3K enzymes are also involved in mobility and migration (Sawyer, Expert Opinion investing. Drugs, 2004, 13, 1-19), PI3K inhibitors are thought to provide a therapeutic benefit via inhibition of tumour cell invasion and metastasis.
The synthesis of small compounds which specifically inhibit, regulate and/or modulate PI3 kinase signal transduction is therefore desirable and an aim of the present invention.
It has been found that the compounds according to the invention and salts thereof have very valuable pharmacological properties while being well tolerated.
It has been found that the compounds according to the invention are inhibitors of the phosphoinositide 3-kinases (PI3Ks).
The compounds according to the invention inhibit protein kinases, in particular PI3K, mTOR and DNA-PK. In addition, they activate Foxo3A translocation.
According to the literature, DNA-PK and mTOR, like the PI3Ks, play an important role in biochemical regulation mechanisms and thus, for example, in the development and spread of cancer diseases.
Thus, other heterocyclic DNA-PK inhibitors are described in WO 2009/130469. mTOR kinase inhibitors for combating cancer are described in WO 2010/062571 A1.
If the phosphoinositide 3-kinase (PI3K) enzyme is inhibited by a compound according to the invention, PI3K is unable to exert its enzymatic, biological and/or pharmacological effects. The compounds according to the invention are therefore suitable for the treatment of autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurodegenerative diseases, allergy, asthma, pancreatitis, multiorgan failure, kidney diseases, blood platelet aggregation, cancer, sperm motility, transplant rejection, graft rejection and lung injuries.
The compounds of the formula I are suitable, in particular, as medicaments for the treatment of autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurodegenerative diseases, allergy, asthma, pancreatitis, multi-organ failure, kidney diseases, blood platelet aggregation, cancer, sperm motility, transplant rejection, graft rejection and lung injuries.
The compounds of the formula I are also suitable, in particular, for the treatment of cancer together with radiotherapy.
According to an embodiment of the present invention, the compounds of the formula (I) are inhibitors of one or more phosphatoinositide 3-kinases (PI3Ks), advantageously phosphatoinositide 3-kinase γ (PI3Kγ), phosphatoinositide 3-kinase α (PI3Kα), phosphatoinositide 3-kinase β (PI3Kβ), and/or phosphatoinositide 3-kinase δ (PI3Kδ).
The compounds of the formula (I) are suitable for the modulation, in particular for the inhibition, of the activity of phosphatoinositide 3-kinases (PI3Ks), advantageously phosphatoinositide 3-kinase (PI3Kα). The compounds according to the invention are therefore also suitable for the treatment of disorders which are promoted by PI3Ks. The treatment includes the modulation—in particular the inhibition or downregulation—of phosphatoinositide 3-kinases.
The compounds according to the invention are preferably used for the preparation of a medicament for the treatment of a disorder selected from multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, lung inflammation, thrombosis or brain infection or inflammation, such as meningitis or encephalitis, Alzheimer's disease, Huntington's disease, CNS trauma, stroke or ischaemic states, cardiovascular diseases, such as atherosclerosis, cardiac hypertrophy, cardiac myocyte dysfunction, hypertension or vasoconstriction.
The compounds of the formula I are preferably suitable for the treatment of autoimmune diseases or inflammatory diseases, such as multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, lung inflammation, thrombosis or brain infection or inflammation, such as meningitis or encephalitis.
The compounds of the formula I are preferably suitable for the treatment of neurodegenerative diseases, such as, inter alia, multiple sclerosis, Alzheimer's disease, Huntington's disease, CNS trauma, stroke or ischaemic states.
The compounds of the formula I are preferably suitable for the treatment of cardiovascular diseases, such as atherosclerosis, cardiac hypertrophy, cardiac myocyte dysfunction, hypertension or vasoconstriction.
The compounds of the formula I are preferably suitable for the treatment of chronic obstructive pulmonary disease, anaphylactic shock fibrosis, psoriasis, allergic diseases, asthma, stroke, ischaemic states, ischemia-reperfusion, blood platelet aggregation or activation, skeletal muscle atrophy or hypertrophy, leukocyte recruitment in cancer tissue, angiogenesis, invasion metastasis, in particular melanoma, Karposi's sarcoma, acute and chronic bacterial and viral infections, sepsis, transplant rejection, graft rejection, glomerulosclerosis, glomerulonephritis, progressive renal fibrosis, endothelial and epithelial injuries in the lung, and lung airway inflammation.
Since the pharmaceutically active compounds of the present invention are active as PI3 kinase inhibitors, in particular the compounds which inhibit pI3Kα, either selectively or together with one or more of PI3Kδ, PI3Kβ and/or PI3Kγ, they have therapeutic utility in the treatment of cancer.
The invention preferably relates to a method for the treatment of cancer in a mammal, including humans, where the cancer is selected from: brain (gliomas), glioblastomas, leukaemias, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast cancer, inflammatory breast cancer, Wilm's tumour, Ewing's sarcoma, rhabdomyosarcoma, ependymoma, medullo-blastoma, colon, head and neck, kidney, lung, liver, melanoma, ovary, pancreas, prostate, sarcoma, osteosarcoma, giant-cell tumour of bone and thyroid.
The invention preferably relates to a method for the treatment of cancer in a mammal, including humans, where the cancer is selected from: lymphoblastic T-cell leukaemia, chronic myelogenous leukaemia, chronic lymphocytic leukaemia, hairy-cell leukaemia, acute lymphoblastic leukaemia, acute myelogenous leukaemia, chronic neutrophilic leukaemia, acute lymphoblastic T-cell leukaemia, plasmacytoma, immunoblastic large cell leukaemia, mantle cell leukaemia, multiple myeloma, megakaryoblastic leukaemia, multiple myeloma, acute megakaryocytic leukaemia, promyelocytic leukaemia and erythro-leukaemia.
The invention preferably relates to a method for the treatment of cancer in a mammal, including humans, where the cancer is selected from malignant lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lymphoblastic T-cell lymphoma, Burkitt's lymphoma and follicular lymphoma.
The invention preferably relates to a method for the treatment of cancer in a mammal, including humans, where the cancer is selected from: neuroblastoma, bladder cancer, urothelial cancer, lung cancer, vulvar cancer, cervical cancer, endometrial cancer, renal cancer, mesothelioma, oesophageal cancer, salivary gland cancer, hepatocellular cancer, bowel cancer, nasopharyngeal cancer, buccal cancer, mouth cancer, GIST (gastrointestinal stromal tumour) and testicular cancer.
The compounds of the formula I can furthermore be used for the isolation and investigation of the activity or expression of PI3 kinase. In addition, they are particularly suitable for use in diagnostic methods for diseases in connection with unregulated or disturbed PI3 kinase activity.
It can be shown that the compounds according to the invention have an anti-proliferative action in vivo in a xenotransplant tumour model. The compounds according to the invention are administered to a patient having a hyperproliferative disease, for example to inhibit tumour growth, to reduce inflammation associated with a lymphoproliferative disease, to inhibit transplant rejection or neurological damage due to tissue repair, etc. The present compounds are suitable for prophylactic or therapeutic purposes. As used herein, the term “treatment” is used to refer to both prevention of diseases and treatment of pre-existing conditions. The prevention of proliferation is achieved by administration of the compounds according to the invention prior to the development of overt disease, for example to prevent the growth of tumours, prevent meta-static growth, diminish restenosis associated with cardiovascular surgery, etc. Alternatively, the compounds are used for the treatment of ongoing diseases by stabilising or improving the clinical symptoms of the patient.
The host or patient can belong to any mammalian species, for example a primate species, particularly humans; rodents, including mice, rats and hamsters; rabbits; horses, cows, dogs, cats, etc. Animal models are of interest for experimental investigations, providing a model for treatment of human disease.
The susceptibility of a particular cell to treatment with the compounds according to the invention can be determined by in vitro tests. Typically, a culture of the cell is combined with a compound according to the invention at various concentrations for a period of time which is sufficient to allow the active agents to induce cell death or to inhibit migration, usually between about one hour and one week. In vitro testing can be carried out using cultivated cells from a biopsy sample. The viable cells remaining after the treatment are then counted.
The dose varies depending on the specific compound used, the specific disease, the patient status, etc. A therapeutic dose is typically sufficient considerably to reduce the undesired cell population in the target tissue while the viability of the patient is maintained. The treatment is generally continued until a considerable reduction has occurred, for example an at least about 50% reduction in the cell burden, and may be continued until essentially no more undesired cells are detected in the body.
For identification of a signal transduction pathway and for detection of inter-actions between various signal transduction pathways, various scientists have developed suitable models or model systems, for example cell culture models (for example Khwaja et al., EMBO, 1997, 16, 2783-93) and models of transgenic animals (for example White et al., Oncogene, 2001, 20, 7064-7072). For the determination of certain stages in the signal transduction cascade, inter-acting compounds can be utilised in order to modulate the signal (for example Stephens et al., Biochemical J., 2000, 351, 95-105). The compounds according to the invention can also be used as reagents for testing kinase-dependent signal transduction pathways in animals and/or cell culture models or in the clinical diseases mentioned in this application.
Measurement of the kinase activity is a technique which is well known to the person skilled in the art. Generic test systems for the determination of the kinase activity using substrates, for example histone (for example Alessi et al., FEBS Lett. 1996, 399, 3, pages 333-338) or the basic myelin protein, are described in the literature (for example Campos-González, R. and Glenney, Jr., J. R. 1992, J. Biol. Chem. 267, page 14535).
For the identification of kinase inhibitors, various assay systems are available. In scintillation proximity assay (Sorg et al., J. of Biomolecular Screening, 2002, 7, 11-19) and flashplate assay, the radioactive phosphorylation of a protein or peptide as substrate with γATP is measured. In the presence of an inhibitory compound, a decreased radioactive signal, or none at all, is detectable. Furthermore, homogeneous time-resolved fluorescence resonance energy transfer (HTR-FRET) and fluorescence polarisation (FP) technologies are suitable as assay methods (Sills et al., J. of Biomolecular Screening, 2002, 191-214). Other non-radioactive ELISA assay methods use specific phospho-antibodies (phospho-ABs). The phospho-AB binds only the phosphorylated substrate. This binding can be detected by chemiluminescence using a second peroxidase-conjugated anti-sheep antibody (Ross et al., 2002, Biochem. J.).